Last class

• Sterilization and disinfection

• Types of sterilization and disinfection
– Physical
– Chemical
– Mechanical
• Controlling mechanisms
Sterilization and disinfection : Overview

2
Principles of diagnostic medical
microbiology
Collection, storage & transport of samples & diagnosis of microbial
infection
Guidelines for specimen collection, storage and transport
Procedures of sample collection, storage and transport
Microbiological laboratory techniques
Microscopic examination
Gram staining
Acid-fast staining
Culture and identification
Biochemical tests
Antimicrobial susceptibility testing
Cell structure
Animal inoculation
Skin tests
Serological techniques
Molecular techniques 4
4
General concepts
– Some infectious diseases are distinctive enough to be identified
clinically.
– Most pathogens, however, can cause a wide spectrum of clinical
syndromes in humans.
– Conversely, a single clinical syndrome may result from infection with
many pathogens
– E.g:- Influenza virus infection,
– causes a wide variety of respiratory syndromes
– cannot be distinguished clinically from those caused by
streptococci, mycoplasmas, or more than 100 other viruses.
– therefore, it is necessary to use microbiologic
laboratory methods to identify a specific etiologic
agent.

– The job of the clinical microbiology laboratory

• test specimens from patients for microorganisms

• provide information about the in vitro activity of

antimicrobial drugs
• The objectives of diagnostic microbiology are
therefore

1. Identification of etiological agent/s that caused disease

2. Appropriate selection of antimicrobial agents for treating

patients

So diagnostic microbiology can Assisting physicians/Clinicians in

the diagnosis and treatment of infection

3. Design a strategy in the control and prevention of infectious

diseases

7
• Manifestations of Infection
– The clinical presentation of an infectious disease
reflects interaction between the host and the
microorganism.
• host immune status and
• microbial virulence factors.
– Signs and symptoms vary according to the site and
severity of infection.
– Diagnosis requires a composite of information,
including
• history,
• physical examination,
• radiographic findings, and
• laboratory data.
• Microbial Causes of Infection
– Infections may be caused by bacteria, viruses, fungi,
and parasites.

– The pathogen may be exogenous or endogenous

(from the normal flora).
• Specimen Selection, Collection, and
Processing
– Specimens are selected on the basis of signs and
symptoms,
– should be representative of the disease process,
– should be collected before administration of
antimicrobial agents.
– The specimen amount and the rapidity of transport
to the laboratory influence the test results.
 • Guidelines for sample collection, storage and
transport
 Proper collection of an appropriate clinical specimen is the first
step in obtaining an accurate laboratory diagnosis of an infectious
disease

 Guidelines for the collection and transportation

available to clinicians in a clearly written format

 The guidelines must emphasize two important aspects:

 Collection of the specimen should be before the administration
of antimicrobial agents
 Prevention of contamination of the specimen with externally
11
present organisms or normal flora of the body.
• The general rules for collection and transportation of
specimens are summarized as:

– Apply strict aseptic techniques throughout the procedure

– Wash hands before and after the collection

– Collect the specimen at the appropriate phase of disease

– representative specimen of the infectious process

– Collect or place the specimen aseptically in a sterile and/or

appropriate container

12
– Ensure that the outside of the specimen container is clean and
uncontaminated

– Close the container tightly so that its contents do not leak

during transportation

– Label and date the container appropriately

– Arrange for immediate transportation of the specimen to the

laboratory

13
• Criteria for rejection of specimens

• Criteria should be developed by a laboratory as in the

following some examples:

– Missing or inadequate identification

– Insufficient quantity

– Specimen collected in an inappropriate container

– Contamination suspected

– Inappropriate transport or storage

– Unknown time delay 14

• Different samples collected for microbiological
laboratory:
– Blood (a drop-20ml ) is required for microbiological
examination of systemic infections

– CSF (3-5 ml) is needed for the diagnosis of any

patient with evidence of meningeal irritation or
affected cerebrum

– Sputum (5-10ml)For investigation of bacterial and

fungal infections of the lower respiratory tract

15
– Urine (1-5ml) for microbes that cause urinary tract
infection

– Stool (1-2ml) Collect fecal specimens for the etiological

diagnosis of acute infectious diarrheas

– Pus and wound secretions for surface wounds

Medical Microbiology lecture guide 16

 Microbiologic Examination

– Direct Examination

– Culture

– Serodiagnosis

– Molecular Technique
 Direct Examination

• Direct examination of specimens frequently

provides the most rapid indication of
microbial infection.

• A variety of microscopic, immunologic, and

hybridization techniques have been
developed for rapid diagnosis
 Direct Examination of Bacteria
• Unstained Preparations
– Wet mounts: sample + Normal saline/KOH
– Hanging drop technique
• Stained Preparations
– Simple staining
– Differential staining
• Gram stain
• Acid fast stain
– Special staining
 Direct Examination of virus

1. Electron Microscopy morphology of virus

particles

2. Light Microscopy histological appearance

inclusion bodies

3. Immunohistochemical staining Antigen

detection
 Direct Examination of fungi
• Microscopic Methods
– The saline mount
• To observe fungal elements
– KOH preparation
• Initial examination of keratinized tissue suspected of fungal
infection
– Staining methods
1. Calcofluor
2. Lacto phenol cotton blue(LPCB)
3. Indian ink preparation
4. Gram staining
5. Giemsa or wright’s stain

6. Gomori Methanamine silver nitrate stain

7. Periodic acid –schiff stain

 Culture
• In many instances, the cause of an infection is
confirmed by isolating and culturing
microorganism
– either in artificial media or in a living host.

• Bacteria (including mycobacteria and

mycoplasmas) and fungi are cultured in either
– liquid (broth) or on solid (agar) artificial media.
 Culture…
• Liquid media provide greater sensitivity for the
isolation of small numbers of microorganisms;

• However, identification of mixed cultures growing in

liquid media requires subculture onto solid media

– so that isolated colonies can be processed

separately for identification
 Culture
Identification of microorganism From culture growth can be made on
the basis of:
– Colony characteristics which includes size, shape, color, odour,
hemolysis, pigment production, swarming

– Observe the turbidity in the case of broth media

– Microscopic examination on unstained and stained preparations

(Gram stain, Acid-fast stain, or other stains)
BIOCHEMICAL TESTS

26
 Biochemical Tests for identification

• These tests have also great role in identifying the type of

species or genus under investigation.
• Identification by biochemical tests is based on:
① Type of enzyme they produce. E.g:- Catalase, Coagulase,
etc
② Ability to ferment specific carbohydrates. E.g:- Lactose
③ Type of their carbon source. E.g:- Mannitol, citrate
④ Type of their byproduct of metabolism. E.g:- Hydrogen
sulfide.
1. Catalase test

This test is used to differentiate those bacteria that produce

the enzyme catalase such as Staphylococci from non

catalase producing bacteria such as Streptococci.

Principle:
– Catalase acts as a catalyst in the breakdown of hydrogen
peroxide to oxygen and water.
– An organism is tested for catalase production by bringing it
in to contact with hydrogen peroxide.
– Bubbles of oxygen are released if the organism is a
catalase producer.
 Catalase test … Cont’d

Figure: Catalase tube test.

Right: Shows a positive test.

Left: Shows a negative test.

Results
• Active bubbling ----- Positive test - Catalase produced
• No release of bubbles ----- Negative test - No catalase produced

Control
• Positive catalase control – Staphylococcus species
• Negative catalase control – Streptococcus species 29
2. Coagulase Test

• Used to differentiate Staphylococcus aureus which produces the

enzyme coagulase, from S.epidermidis and S.saprophyticus which
do not produce coagulase.

• Coagulase causes plasma to clot by converting fibrinogen to fibrin.

Clotting in the tube  S.aureus

No clotting Negative test (CONS)
 Coagulase Test…Cont’d

Results
• Clotting in the tube……….S.aureus
• No clotting ……………… Negative test
Note: There should be no clotting in the negative control tube.

31
 3. Oxidase test/Cytochrome oxidase test

• The oxidase test is used to detect bacteria that produce the

enzyme cytochrome oxidase which catalyze oxidation of
reduced cytochrome by oxygen molecule

• When the organism is oxidase-producing, the oxidase reagent

(Phenylenediamine) will be oxidized to a deep purple color.

Result
Blue – purple color …..positive Oxidase test
(With in 10 sec)
No blue – Purple color …Negative Oxidase test
(With in 10 sec)
 Oxidase test cont’n…
• It assist in the identification of
– Pseudomonas,
– Neisseria,
– Vibrio,
– Brucella, and
– pasteurella species, which are oxidase positive.

Controls
– Positive oxidase control: Pseudomonas
aeruginosa
– Negative oxidase control: Escherichia coli
 4. Fermentation of carbohydrates

• Carbohydrates are complex chemical substrates that serve

as energy sources when broken down by bacteria and
other cells.

• Facultative anaerobic and anaerobic bacteria are

capable of fermentation, an anaerobic process during
which carbohydrates are broken down for energy
production.
• When carbohydrates are fermented as a result of
bacterial enzymes, the following fermentation end
products may be produced:
 Acid end products, or
 Acid and gas end products.
• If carbohydrate is fermented by the bacterium,
– acid end products will be produced
– which lowers the pH,
– causing the pH indicator to change color (phenol red turns
yellow).
• If gas is produced along with the acid, it collects in the Durham
tube as a gas bubble

• If the carbohydrate is not fermented, no acid or gas will be

produced and the phenol red will remain red.

A B C D E F G
Reading assignment
• Remaining important biochemical tests
– Urease test
– Indole
– Citrate utilization test
– Bile solubility test PRINCIPLE
– DNase test
– Litmus milk decolorization test AND
– MRVP (methyl red-Vogues Proskauer) test
– VP (Vogues Proskauer) test INTERPRETATION ?
– Triple sugar Iron
– Kligler Iron Agar (KIA)
– Nitrate reduction test
– Motility-Indole-Urea (MIU)
– API 20E
 Antimicrobial sensitivity test (AST)
• Used to measure the ability of the drug to inhibit or kill
pathogens in vitro by determining the MIC (minimum inhibitory
concentration).

I.e. it is used to select effective antimicrobial drugs.

• The objective of AST is to:

– Guide the clinician/s in the selection of the most appropriate
antimicrobial agent/s
– Provide clinician/s with an alternative agents to which the organism
is susceptible
– Detect and control the spread of drug resistant organisms
• Generally, drug Sensitivity (susceptibility) testing (DST)
can be performed:
For organisms with variable antibiotic sensitivity (un
predictable sensitivity) E.g. Shigella spp
For non responding patients after taking adequate
therapy.
For patients whose immune system is depressed
For relapsing cases (reappearance of disease)
 Methods of Antimicrobial Susceptibility Testing

• Two types of antimicrobial susceptibility testing

methods:

1. Disk Diffusion method

– Kirby-Bauer disc diffusion method

2. Dilution method

– Broth dilution

– Agar Dilution
1. Disc Diffusion Test:

• Appropriate plated medium is inoculated with a standardized

inoculum of a pathogen

• Filter paper disks with standardized concentrations of drugs are

placed on the agar; incubated at 37°C for 18 hrs

• Antimicrobial drug diffuses and produces a zone of inhibition

• Diameter of zones of inhibition are measured around disk

Antibiotic susceptibility testing

42
• Disk diffusion method
Disk diffusion method

Measuring the diameter of zones of inhibition in mm.

47
 Interpretation of disc diffusion (Kirby-Bauer Test)

The Kirby-Bauer Test is used to determine the effectiveness of a drug by

measuring the zone of inhibition. 48
Clinical Evaluation
– The result of AST pattern could be either of the following

• Susceptible: respond to treatment with that antimicrobial

agent in normal recommended doses.

• Resistant: will not respond to treatment with that drug to

which it is resistant irrespective of dose

• Intermediate: higher dose may be required to ensure the

efficacy of the antimicrobial agents

N.B: In some cases, massive doses could be given to inhibit

microbes if the antimicrobial is not toxic like penicillins
49
2. Dilution Method:
• Concentrations similar to those achievable in tissues with
normal doses of the drug are inoculated with an organism
and incubated at 37° C for 18 hours.
– Dilutions of the antimicrobials prepared in tubes and
– a fixed bacterial inoculum is added;
– Turbidity indicates growth
– Then determine the MIC and MBC
• The end point with out growth is taken as that smallest amount of
antimicrobial agent required to inhibit the growth of the test
bacteria or to kill the test bacterial

– The lowest concentration of the drug that inhibits growth of

the organism = MIC (minimum inhibitory concentration)

– The concentration required to kill bacteria = Minimum

bactericidal concentration (MBC) and it is found by sub-
culturing from the previous tubes with no growth in antibiotic
free media.
N.B: the MIC or MBC value is then compared
with known concentration of drug
obtainable in the serum or other body
fluids, and then the likely clinical
response can be assessed.
FIGURE: Broth dilution susceptibility test.
 Microbiological laboratory techniques..
• Serological techniques

– Microbial infections commonly elicit immune responses

• against one or more antigens of the infectious agent

– Serologic diagnosis targets the humoral immune response

– Antibodies of the IgM class appear initially and are followed by

IgG antibodies

– The IgM antibodies disappear in several weeks, whereas the

IgG antibodies persist for many years

54
 Microbiological laboratory techniques

• Serological techniques…

– Some of the examples of serological techniques include

• Neutralization test (Nt)

• Hemagglutination inhibition test (HIT)

• Compliment fixation test (CFT)

• Immunoflorescence (IF)

• Immunodiffusion (IDF)

• Enzyme linked immunosorbent assay (ELISA) etc.

55
• Molecular techniques
– modern and highly sensitive diagnostic tests

– based on amplifying of nucleic acids of the microorganisms

– Most commonly used Molecular techniques is Polymerase

chain reaction (PCR)

• Transcription-mediated amplification (TMA)

• Ligase chain reaction (LCR)

56
 General summary
1. Obtain a specimen from infected site
2. Non stained and Stain the specimen using
appropriate procedure
3. Culture the specimen on the appropriate
media
4. Identify the organism using the appropriate
tests
5. Perform antibiotic susceptibility test

57
End for Module One !!!